Secondary structures of narcissus mosaic virus coat protein.

Circular dichroism (CD) measurements on the coat protein of narcissus mosaic virus particles show that the dominant secondary structure is the alpha-helix, with a 45 (+/- 2)% content. The beta-sheet content is much lower at 5 (+/- 3)%. Both values are essentially the same as those found in potato virus X coat protein. The CD results are used to assess the results of secondary structure prediction methods.

cDNA cloning and nucleotide sequence of the wheat streak mosaic virus capsid protein gene.

The 3'-terminal region of wheat streak mosaic virus (WSMV) genomic RNA was cloned and a cDNA sequence of 1809 nucleotides upstream of the poly(A) tract was determined. The sequence contains a single open reading frame of 1662 nucleotides and a 3' untranslated region of 147 nucleotides. Translation products from WSMV RNA and WSMV cDNA transcripts were immunoprecipitated by WSMV capsid protein antiserum, indicating that the 3'-terminal region of WSMV RNA encodes the capsid protein. Five potential N-terminal capsid protein protease cleavage sites were identified, which would yield proteins ranging from 31.7K to 46.8K. Alignment of the deduced amino acid sequence of the WSMV capsid protein with those of other potyviruses showed significant, but limited, identity as compared to the alignment of two or more aphid-transmitted potyviruses. Although WSMV has characteristics distinct from potyviruses, because of its particle morphology, translation strategy apparently based on polyprotein processing, the ability to form cytoplasmic cylindrical inclusions and the degree of capsid protein homology with aphid-transmitted potyviruses, it should be considered a member of the potyvirus group.

Ubiquitinated conjugates are found in preparations of several plant viruses.

Recently D.D. Dunigan, R.G. Dietzgen, J.E. Schoelz, and M. Zaitlin (Virology 165, 310-312, 1988) demonstrated that a small proportion of the subunits of tobacco mosaic virus particles were conjugated with the small protein ubiquitin. We have now detected ubiquitinated conjugates in immunoblots of virion preparations of several other plant viruses, using anti-human ubiquitin antiserum. Based on their polyacrylamide gel migrations, plant virus-associated ubiquitin-immunoreactive proteins were considered to be possible virus structural protein-ubiquitin conjugates of the following viruses: barley stripe mosaic, brome mosaic, cowpea mosaic (two proteins), cowpea severe mosaic (two proteins), and satellite panicum mosaic. Ubiquitinated conjugates were not detected in immunoblots of preparations of cucumber mosaic virus and Cymbidium mosaic virus. The significance of ubiquitinated viral proteins remains to be determined.

Purification of plant viruses and virus coat proteins by high performance liquid chromatography.

High performance liquid chromatography (HPLC) gel filtration has been successfully applied in the purification of elongated and isometric plant viruses. Two different approaches have been tested. In one approach, semi-purified virus particles were dissociated with lithium chloride and the released coat proteins purified by HPLC gel filtration. The purified coat protein was highly immunogenic and gave rise to very specific antisera reacting with intact virus particles as well as with SDS-denatured coat protein monomers. This method is generally applicable only for elongated viruses since many isometric viruses are not dissociated by the lithium chloride treatment. The second approach consisted in gel filtration of native, undissociated virus particles and could be used both with elongated and isometric viruses. Both methods were fast and simple to perform and removed all or most of the contaminating plant proteins as judged by sodium dodecylsulphate gel electrophoresis followed by silver staining or by immunoblotting with antiserum against healthy plant extracts. With both methods the recovery of virus coat protein was about 30% on average.

The primary structure of papaya mosaic virus coat protein: a revision.

The presence of an acetyl blocking group at the N-terminus of the coat protein of papaya mosaic virus has been identified by FAB mass spectrometry. Furthermore, we have found that the N-terminal sequence of the protein is four amino-acid residues (AC-Ser-Lys-Ser-Ser-) longer than that previously reported, while Glu instead of Gln is the C-terminal residue. The present paper shows that PMV-protein is made up of 215 amino acid residues, with a molecular mass of 22,960 Da.

Biochemical and biophysical analysis of pseudoknot-containing RNA fragments. Melting studies and NMR spectroscopy.

Three overlapping RNA fragments containing the pseudoknot, as found in the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA, have been isolated and purified. Site-directed cleavage of TYMV RNA by RNase H, followed by ammonium sulphate precipitation and ion-exchange HPLC, yielded a pure preparation of a 3'-terminal, 112-nucleotide TYMV RNA fragment. Transcription of TYMV cDNA by T7 RNA polymerase, resulted in the isolation of an 88-nucleotide fragment. Finally, a 44-nucleotide fragment containing the TYMV RNA pseudoknot and strongly resembling the aminoacyl acceptor arm of the viral RNA was also synthesised using T7 RNA polymerase. The three fragments were isolated in milligram amounts and used for biochemical structure mapping, ultraviolet melting studies and NMR spectroscopy. Chemical modification with diethyl pyrocarbonate and sodium bisulphite and enzymatic digestion with RNase T1 confirmed the presence of the pseudoknot in the 44-nucleotide fragment. Also the analogue of the T-stem and T-loop of the tRNA-like structure of TYMV RNA was found. The results of modification at various temperatures in Mg2+-containing buffers were in general agreement with optical melting studies. Ultraviolet melting analysis of the longer fragments revealed their greater complexity and the results appear similar to those obtained for some tRNA species. To obtain direct biophysical evidence for base-pairing and stacking interactions in the pseudoknot, NMR studies were initiated. The first proton-NMR spectra ever obtained for plant viral RNA fragments are presented. NMR spectra were recorded at various buffer conditions and at various temperatures. The spectra for the 112-nucleotide and 88-nucleotide fragment are too complicated to be solved at present. In the case of the 44-nucleotide fragment, however, the imino proton resonances are well separated and this system turns out to be most promising for structural studies.

Preparation of soluble, biologically active alfalfa mosaic virus coat protein and its CaCl2-induced degradation.

A method for the preparation of soluble protein from five biologically distinct alfalfa mosaic virus (AMV) isolates is described. Highly purified AMV was dissociated with 1 M CaCl2 in 10 mM sodium acetate, pH 6.0, and the precipitated RNA was removed by centrifugation. The protein was dialysed against 10 mM sodium acetate, pH 6.0, containing 0.1 M CaCl2. If the salt concentration was reduced further, proteins from some AMV isolates precipitated. Proteins prepared by this method were shown to be immunoreactive and to activate the infectivity of the AMV genome. However, during prolonged exposure of the protein to buffers containing 0.1 M CaCl2, it undergoes slow proteolysis thereby losing its ability to activate the AMV genome but not its immunoreactivity.

Protein-RNA interactions in an icosahedral virus at 3.0 A resolution.

Nearly 20 percent of the packaged RNA in bean-pod mottle virus (BPMV) binds to the capsid interior in a symmetric fashion and is clearly visible in the electron density map. The RNA displaying icosahedral symmetry is single-stranded with well-defined polarity and stereochemical properties. Interactions with protein are dominated by nonbonding forces with few specific contacts. The tertiary and quaternary structures of the BPMV capsid proteins are similar to those observed in animal picornaviruses, supporting the close relation between plant comoviruses and animal picornaviruses established by previous biological studies.

Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions.

Temperature-gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein-nucleic acid interactions. A linear and highly reproducible temperature-gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature-gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand-separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double-stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (-)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch-formation during the renaturation of (+)- and (-)strands from different clones. Examples are given for different viroid clones and HIV-clones from one and the same patient. In another example, clones with point mutations from site-directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the repressor and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.

Relationship of lychnis ringspot virus to barley stripe mosaic virus and poa semilatent virus.

Barley stripe mosaic virus (BSMV), poa semilatent virus (PSLV), and lychnis ringspot virus (LRSV) have previously been assigned to the hordeivirus group because of similarities in their particle morphology, physicochemical properties and serological analyses. However, the serological relationships of the three viruses have not been determined by direct comparison. The present study evaluated the relatedness of these viruses by Western and dot immunoblotting and by nucleic acid hybridizations. Serological analyses of the coat proteins separated by gel electrophoresis and of intact virus particles bound to nitrocellulose membranes revealed that BSMV and PSLV are distantly related, but that they are more closely related to each other than to LRSV. The genomic RNAs of the viruses failed to cross-hybridize in northern hybridization tests conducted at different temperatures. These comparisons showed that BSMV, PSLV and LRSV are distinct viruses with little nucleotide sequence relatedness. Thus our data provide additional support for their inclusion as separate members of the hordeivirus group.

Characterization of bean mild mosaic virus: particle morphology, composition and RNA cell-free translation.

Virions of bean mild mosaic virus (BMMV) are built of 180 subunits of a single protein species of MW 40 x 10(3) [coat protein CP], packed into a T = 3 surface lattice. The capsomers on the five-fold symmetry axes protrude 2-3 nm from the particle surface. The virions encapsidate genome-size [approximately 4,200 nucleotides (nt)] as well as some heterogeneous RNAs of subgenomic size approximately 1,000-2,000 nt. In cell-free systems from Krebs-2 ascites cell extracts and rabbit reticulocyte lysates, genome-size RNA directed the synthesis predominantly of two polypeptides of MW 27 x 10(3) and 79 x 10(3) while the third major BMMV-specific polypeptide (MW 40 x 10(3), putative CP) seemed to be encoded by a shorter messenger RNA. The 'cap' analogue, m7GDP, partially inhibited BMMV RNA in vitro translation, suggesting that at least part of the BMMV-specific RNAs are capped. Oligo (dT)-cellulose column chromatography data suggested that poly(A)-tracts are absent from the BMMV genome. The data obtained confirm the previous classification of BMMV within the carmovirus group.

Characterization of cucumber mosaic virus. I. Molecular heterogeneity mapping of RNA 3 in eight CMV strains.

RNAs from 13 strains of cucumber mosaic virus (CMV) were divided into two groups on the basis of their ability to hybridize to cDNA of either Fny-CMV RNA or WL-CMV RNA. The extent of the cross-hybridization within one of these groups was analyzed by an RNA protection assay. A cDNA clone of RNA 3 of the Fny strain of CMV was placed in a transcription vector between bacterial promoters T3 and T7. Labeled, minus-sense RNA transcripts prepared from all or part of the cDNA to RNA 3 of Fny-CMV were annealed to the genomic RNA of each of a number of cucumoviruses and digested with RNases. The patterns of RNA fragments protected from digestion were specific for each CMV strain and revealed the extent and location of heterogeneity among the viruses as well as within the Fny-CMV natural population. This approach will allow the differences in host range and disease processes to be correlated with variations in genomic RNAs.

A beneficial effect of trypsin on the purification of turnip mosaic virus (TuMV) and other potyviruses.

The effects of treatment with trypsin during the purification of turnip mosaic virus (TuMV), on virus yield, infectivity and integrity of virus coat protein were examined. Trypsin increased yield markedly, and at a low concentration, increased infectivity. These effects were probably due to reduced aggregation of virus particles. At higher concentrations of trypsin, there was some degradation of virus coat protein, and infectivity was reduced. Treatment with trypsin at the optimum concentration can significantly improve purification of TuMV; more limited experiments suggest that it can also be applied to other potyviruses.

Analysis and application of substrate hydrolysis rates in indirect ELISA of a purified plant virus.

The transient colorimetric signal in a microtiter plate is used to quantify a purified plant virus, cowpea mosaic virus (CPMV), over five concentration decades in a single plate. The method involves the coating of the polystyrene microtiter plate wells directly with the CPMV antigen, followed by incubation with a rabbit-derived CPMV-specific antibody, and lastly by incubation with a commercially available antibody against rabbit immunoglobulin which has been pre-labeled with alkaline phosphatase. The rate of p-nitrophenylphosphate hydrolysis, both non-specific and that which was catalyzed by this enzyme, was measured spectrophotometrically at 405 nm. Enzyme-catalyzed hydrolysis rates followed first order kinetics at all antigen coating concentrations, and the 1 degree rate constants, which ranged from 2 X 10(-6) min-1 to 1 X 10(-3) min-1, were found to increase with increasing antigen concentration.

The dynamical structure of the RNA in alfalfa mosaic virus studied by 31P-nuclear magnetic resonance.

The structure of the viral RNA in alfalfa mosaic virus (AlMV) was investigated by means of 31P-nuclear magnetic resonance (NMR). It was found that the 31P-NMR line width of AlMV Top a particles is significantly smaller than that of the larger Bottom particles. At low temperatures, the totational correlation time of the 31P nuclei essentially equals the tumbling rate of the virus particle, indicating that the RNA is contained rigidly inside the virion. At more elevated temperatures, the NMR line width sharpens more than expected on the basis of viscosity changes and the RNA exhibits internal mobility. The occurrence of internal mobility is paralleled by an increased internal mobility of the N-terminal part of the coat protein, as could be observed by 1H-NMR spectroscopy. The influence of EDTA on the 31P-NMR line width appeared to be negligible, which is in agreement with the idea that AlMV does not 'swell' like several other RNA-containing plant viruses.

The isolation of the two electrophoretic forms of cowpea mosaic virus using fast protein liquid chromatography.

This report describes the first time entire viral capsids have been purified using fast protein liquid chromatography (FPLC) techniques. The FPLC is used here to separate the two electrophoretic forms of cowpea mosaic virus. The capsid forms are shown to be separated by the Mono-Q column without damaging the capsids.

Electron microscopic mapping of single-stranded discontinuities in cauliflower mosaic virus DNA by means of the biotin-avidin technique.

Biotin-labeled deoxyuridine triphosphate has been used to label the three natural single-stranded discontinuities of Cauliflower Mosaic Virus DNA. The presence of biotin permitted the direct visualization and mapping of the discontinuities by electron microscopy, using ferritin-labeled avidin.

Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses.

Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex.

1H, 13C, and 31P nuclear magnetic resonance studies of cowpea mosaic virus: detection and exchange of polyamines and dynamics of the RNA.

1H and 13C NMR studies on cowpea mosaic virus (CpMV) revealed that polyamines are present in the middle (M) and upper bottom (BU) components obtained by CsCl density gradient centrifugation but not in the top (T) component; the lower bottom (BL) component contains trace amounts of polyamine. Dialysis of the BL component against spermidine led to incorporation of spermidine which gave rise to NMR peaks very similar to those observed with the natural M and BU components. NMR results conclusively demonstrate that polyamines in the M and BU components of CpMV are exchangeable with cesium ions and the exchange process is pH dependent. They also provide experimental support for the hypothesis that the BU to BL conversion results from the displacement of polyamines and possibly other natural counter ions of the RNA by cesium ions [G. Bruening, (1977), In "Comprehensive Virology" (H. Fraenkel-Conrat and R. R. Wagner, eds.), Vol. 11, pp. 55-141. Plenum, New York]. No sharp peaks, attributable to mobile amino acid side chains, were seen in spectra of an intact CpMV particle or its empty protein shell (T component). 31P NMR spin-lattice relaxation time and nuclear Overhauser effect parameters, which are sensitive to high-frequency motions, suggest that the RNA and, when present, the bound polyamine undergo internal motions with correlation times in the nanosecond range.